Role of Mef2D in inflammation

The immune system is essential in order to protect from infection and to mediate tissue repair. Despite these important functions the immune system, when not correctly regulated, can have harmful effects. Following infection, pathogens are detected by cells in the immune system, which then trigger an inflammatory reaction. This process results in the recruitment and activation of further immune cells and is designed to kill the pathogen. Once the pathogen is cleared then the immune system is deactivated, a process referred to as resolution. While inflammation is a necessary process to deal with infection, a side effect of this process is unwanted collateral damage to healthy cells in the body. To minimize this effect, inflammation is a tightly controlled process and subject to a range of both positive and negative feedback mechanisms to both prevent uncontrolled inflammation and promote its resolution once pathogens are killed. Co-ordination of immune cells during inflammation is achieved via the production of a range of pro- and anti-inflammatory cytokines, small proteins that control the function of immune cells. Due to their importance in regulating the immune system, the production of these cytokines is tightly regulated and failure of this regulation has serious consequences. For example, failure to limit inflammation during infection can lead to a uncontrolled production of pro-inflammatory cytokines that persists even if the infection is controlled. These 'cytokine storms' can lead to septic shock and are very difficult to treat and still result in a high rate of mortality. An inability to promote the resolution of inflammation or a breakdown in immune tolerance results in chronic inflammation, a condition that underlies pathology in a range of diseases including autoimmune conditions, diabetes and cardiovascular disease.
Drugs that modulate the production or function of cytokines to inactivate the immune system therefore have potential for the treatment of a range of conditions. Understanding the intracellular mechanisms that regulate cytokine production and how this impacts on the immune system is an important first step in selecting targets that can be used for drug development.
We are interested in how the production of an anti-inflammatory cytokine, IL-10, is controlled. IL-10 is a key cytokine in limiting inflammation and promoting resolution. This is illustrated by the finding that mutations that either inactivate the IL-10 gene or block IL-10 function result in the development of colitis in both mice and humans due to a deregulated immune response to the gut flora. In addition loss of IL-10 function has been shown to sensitize mice to pathology in a large range on immune models. In our previous work, we have looked at how transcription of the IL-10 gene is regulated and started to unravel the intracellular pathways that control its expression. Recently we have found that in macrophages, an important cell type in innate immunity, IL-10 production is inhibited by Mef2D, a protein that was not previously established to play a role in macrophage function. Mef2D is a transcription factor that can bind to DNA and regulate the transcription of specific genes and a potential site for Mef2D exists in the IL-10 gene. In the proposed work, we will determine the molecular mechanism by which Mef2D regulates IL-10 in macrophages and the signaling pathways that control Mef2D function. We will also determine if Mef2D plays a similar role in other cell types in the immune system that produce IL-10. Finally we will determine what the effect blocking Mef2D function in vivo has on IL-10 dependent models of immune disease.
Together these experiments will help delineate a new pathway that controls the production of IL-10. This in turn will suggest novel ways in which IL-10 production could be targeted by drugs designed to limit inflammation.

The aim of this work is to determine the roles that Mef2D plays in regulating the immune system. Mef2D is a transcription factor and our preliminary data indicate that it represses TLR induced IL-10 production in macrophages. Knockout of Mef2D increases IL-10 production by TLR4 stimulated bone marrow derived macrophages and as a result represses TNF, IL-6 and IL-12 production. In vivo IL-10 is a key anti-inflammatory cytokine and is implicated in several pathologies including endotoxic shock, infection, psoriasis, inflammatory bowel disease and arthritis. We will therefore examine the mechanism by which Mef2D acts in macrophages, determine if its role is restricted to macrophages and examine the consequence of loss of Mef2D function in disease models.
For the first aim we will make use of primary bone marrow derived macrophages to study the regulation of Mef2D by cell signaling using established biochemical and cell biological techniques. In addition we will use RNAseq approaches to get a genome wide picture of the role of Mef2D in TLR induced transcription in macrophages. We will combine this with ChIP experiments to demonstrate Mef2D localization to its target promoters.
To determine if Mef2D functions in a similar manner in other immune cells we will isolate dendritic cells, T cells and B cells from wild type and Mef2d knockout mice to determine the contribution of Mef2D to the induction of IL-10 in these cells downstream of appropriate stimuli. In parallel we will also examine the role of Mef2D in these cells for the transcription of other target genes that we identify in our BMDM studies.
Finally we will determine the role of Mef2D in selected immune disease models using our knockout mice. Initially we will look at models of endotoxic shock and toxic contact eczema, as these models are known to be influenced by macrophage derived IL-10. Depending on what roles we find for Mef2D in other cells types this list may be expanded or modified as appropriate.

The research project will determine the roles that the transcription factor Mef2D plays in macrophage function and in the regulation of IL-10, a key anti-inflammatory cytokine that impacts on the production of many pro-inflammatory mediators. This will have an impact on our understanding of how cellular signaling pathways regulate the immune system and how they contribute to pathology when this does not occur correctly. This work will therefore inform our understanding of both how the immune system responds to infection and its role in disease. These are important questions. Infectious disease remains a major health risk, especially with the emergence of new bacterial and fungal strains that are resistant to current therapies. Systemic bacterial and fungal infections can result in the development of sepsis that is accompanied by overproduction of cytokines that have a major influence on pathology. Sepsis still has a high mortality rate and is a major cause of death in intensive care units. Deregulation of cytokine production is also a major cause of pathology in autoimmune conditions. Autoimmune disease covers a range of related conditions, which combined represent a major health issue. Together autoimmune conditions have a similar prevalence and health care costs to cardiovascular disease or cancer. Current therapies for autoimmunity rely on immunosuppression and while they arrest the development of the disease they do not provide a cure to the underlying causes or reverse the existing tissue damage. As a result long-term treatment is often required, which frequently leads to complications associated with the side effects of the drugs. The discovery of new drugs to treat these conditions is therefore a major target for the pharmaceutical industry.

The development of new drugs however requires a detailed knowledge of how the immune system works in order to select the best pathways or proteins to target, and our work should have a significant impact in this area. We are in a good position to exploit these findings as my group forms part of the Division of Signal Transduction in Dundee, which represents a major investment by 6 major pharmaceutical companies (GSK, Merck Serono, Pfizer, Astra Zeneca, Boehringer Ingelheim and Janssen Pharmaceutica) into academic research carried out in 17 groups in Dundee. The DSTT consortium also provides us with a way of translating our research; our previous studies have already generated interest with the companies involved and led to the initiation of drug development programs against two of the protein kinases we are currently interested in.

Finally we will also continue to play an active role in informing the public about biomedical research and its importance. To do this we will continue our participation in science festivals in Dundee and Edinburgh that are focused on engaging children in science. We will also seek to expand this work via participation in the STEM Ambassador program, which facilitates the interaction between researchers and science programs in schools. My group also receives funding from Arthritis Research UK, and we have had several meetings with the fundraising division of this charity to talk to current and potential donors about the type of work we undertake and what its benefits are. This is something we are keen to continue with in the future.